Chromatographic and electrophoretic separation of chemicals using electrically conductive polymers

ABSTRACT

A separation system comprising a stationary phase, a double stranded conductive polymer system contacting the stationary phase and means to place a mobile phase carrying a component to be separated from the mobile into contacting relationship with the polymer system whereby the component is captured by the polymer. The double stranded polymer is comprised of a conjugated polymer and a polyelectrolyte.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/175,700, filed Jan. 12, 2000.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to the synthesis and use of doublestranded polymers to separate chemical mixtures.

[0004] 2. Description of the Related Art

[0005] The ability to separate chemical mixtures into their individualcomponents is a billion dollar industry. Speed, resolution, efficiency,reproducibility, repeatability and low cost are all factors thatdetermine a good separation method. Current methods to separate chemicalmixtures include chromatography and electrophoresis. These techniquesare essential for clinical analysis, for biotechnology, environmentalanalysis, and for drug development. Chemical separation primarilyentails the chemical/physical interaction of a stationary surface (astationary phase) with chemicals (in a mobile phase) that flow by thesurface of the stationary phase.

[0006] Chromatography achieves the separation of chemical mixtures bythe use of a mobile phase and a stationary phase. The mixture isinjected into the mobile phase and the mobile phase then flows over thestationary phase. The different interactions of the individualcomponents with this combination of phases creates a separation. If aneutral component has a polar character then it will be retained longestby a polar stationary phase. Neutral non-polar solutes will be retainedbest by a non-polar stationary phase. The mobile phase can be liquid, agas or a supercritical fluid. The stationary phase can be a packed orwall coated standard or capillary column. The type of mobile phasedetermines the name of the method, e.g. liquid chromatography, gaschromatography . . . etc.

[0007] A conductive polymer, namely single strand polyaniline, has beencoated on a glassy carbon stationary phase to achieve charge controlledchromatography. The coating allowed the number of ion exchange sites inthe stationary phase to be controlled instead of being fixed as in ionchromatography and inorganic anions and organic acids were separated.However, a drawback of coating the single strand polyaniline on thestationary phase is that the single strand polyaniline does not have awide enough pH range to be usable.

[0008] Electrophoresis creates separations of charged molecules.Electrophoresis is principally used for the separation of biologicalmolecules and remains a standard biological tool. Charged molecules canbe separated in slab gels by the application of an electric field.Capillary electrophoresis separates charged molecules according to theirelectrophoretic mobilities in an electric field. Generally, theseparation compartment is a narrow fused silica capillary filled with anelectrolyte solution. The electric field is applied with an externalvoltage source between two electrodes in small vials in contact with theelectrolyte solution at both ends of the capillary. The sample isintroduced either hydrostatically or electro migration as a narrow zoneat one end of the capillary. Typically, UV detection takes place at theother end of the capillary.

[0009] Fused silica is the typical capillary material used in capillaryelectrophoresis because it is inexpensive, easy to fabricate intocapillaries with internal diameters in the 10-300 μm ranges, possessesoptical transparency for both UV and visible spectrums, is mechanicallystrong and is flexible when coated with polyimide. However, the materialproperties of fused silica presents some drawbacks when used incapillary elecrophoresis. For example, the surface silanol groups of thefused silica behave as a weak acid, ionizing in water, with a broadtitration curve in the pH 3.9 to 9 region. These surface anionic groupsinduce both electro osmotic flow (EOF) and solute wall interactions tooccur. Solute wall interactions typically occur with cationic proteinsthat electrostatically bind to the silica. Reversible interactionsbetween such analytes and the capillary surface worsen the separationprofile, broadening the peaks and decreasing reproducibility, whileirreversible interactions can destroy the flow profile entirely.Attempts to reduce these interactions include the use of extreme pHbuffers (very high and very low), the use of additives and modificationof the capillary surface.

[0010] Several coatings have been applied to the surfaces of a capillaryto address the drawbacks associated with the use of fused silica as thecapillary material in capillary electrophoresis. For example,nonconducting polymers have been adsorbed to the surface of a fusedsilica capillary. However, the prior art polymeric coatings that havebeen adsorbed to the surface of a fused silica capillary are typicallyunstable.

[0011] The present invention provides a coating for the stationary phaseof a chemical separation system, the coating comprising a doublestranded conductive polymer that is efficient, effective and overcomesthe drawbacks associated with existing chemical separation systems.

BRIEF SUMMARY OF THE INVENTION

[0012] Broadly, the invention comprises a double stranded conductivepolymer functioning as the stationary phase of a chemical and/orbiological separation system. The double stranded conductive polymerprovides controllable interactions between the polymer system of thestationary phase and the chemicals and/or biological analyte in acarrier stream. The invention further comprises a chemical separationsystem comprised of stationary phase comprising a double strandedpolymer.

[0013] The double stranded conductive polymer used in the inventioncomprises a linear strand of polyaniline and a linear strand of apolyelectrolyte twisted together to form a macro-molecule. Thepolyaniline strand can be modified to predictably change itshydrophobicity and/or its color when the pH and/or the electrochemicalpotential within the separation system is changed. The modification ofthe polyaniline strand controls the analyte-surface interactions toimprove the separation. The linear strand of polyelectrolyte providesthe properties suitable for non-aggressive interactions with the analyteor carrier stream.

[0014] The double stranded conductive polymers can be used as a part ofthe stationary phase in a chromatographic column, as a coating on theinner surface of a capillary for separation by capillaryelectrophoresis, as part of a filtration membrane, as a component in gelelectrophoresis and/or coated on or admixed with particulate materialpacked in a column or the like. The double stranded conductive polymerhas the chemical structure that is suitable for selective interactionwith molecules dissolved in a carrier fluid that flows by the polymer toeffect chemical separation of the components in the mixture.

[0015] The double stranded conductive polymers used for chemicalseparation belong to a class of polymers comprising a molecular complexof two strands of polymers: (1) a π-conjugated polymer such aspolyaniline, plypyrrole, polythiophen, poly(phenylene vinylene), etc.and (2) a polyelectrolyte such as poly(acrylic acid),poly(methylvinylether-co-maleic acid), poly(butadiene-co-maleic acid),poly(vinylsulfonic acid), poly(styrenesulfonic acid), poly(methacrylicacid), poly(L-glutamic acid), poly(L-Asparic acid), etc. The two strandsof the molecular complex are bonded non-covalently for most of theapplications, although crosslinking between the two strands is alsopossible. The synthetic process (the template-guided synthesis) allowsthe control of solubility, conformation, and the morphology of thepolymer and thus provides advantageous properties for chemicalseparation applications. The two strands of polymers in the molecularcomplex are likely to be non-covalently bonded in a side-by-sidearrangement thus they are referred to as double-stranded polymers,although the actual structure of the complex could be somewhat random.

[0016] A double stranded conductive polymer was coated on the innersuface of a glass capillary in a capillary electrophoretic separationapparatus. The electro osmotic flow (EOF) carried the chemical mixturethrough the capillary. Due to the influence of the π-conjugated polymerscoated on the capillary wall, the different types of molecules in themobile phase are separated by their difference in elution time. Thepresent invention embodies coatings for improving the analysis oforganic and inorganic species by chromatographic and electrophoretictechniques. These experiments demonstrate the beneficial molecularinteraction between the stationary and the mobile phases. The samemolecular interactions can be used for liquid chromatography, HPLC, ofthin-layer chromatography for chemical analysis. The double strandedconductive polymers are also useful for large-scale separation ofchemicals or drugs when it is used as a component in preparative-scalechromatography or as part of a membrane for selective filtration ofchemicals.

[0017] The double stranded polymer used for the preferred embodimentcomprises two components: (1) a polyaniline molecule, and (2) apolyanion. These two strands of polymers are bonded by non-covalentintermolecular interactions to form a stable molecular complex. Examplesof the polyanion in the polymeric complexes are poly(stryrenesulfonicacid), poly(acrylic acid), poly(methacrylic acid),poly(2-acryamido-2-methyl-1-propenesulfonic acid), andpoly(methylacrylate-co-acrylic acid), poly(butadiene-co-maleic acid),poly(glutamic acid), poly(aspartic acid), etc.

[0018] Another advantage of using the double stranded conductivepolymers in a chemical separation system is the relative ease infunctionalizing the polymer to adjust material properties to meet thedemand for practical applications. The double stranded conductivepolymers are synthesized to be soluble in water, or soluble in organicsolvents, or suspended in latex to satisfy the demands of coatingsapplications. Certain functional groups of the double strandedconductive polymer provide strong adhesion to metals and other polymers,an advantageous property for coatings application for the stationaryphase in the separation system.

[0019] The double-strand conductive polymers are synthesized by a methodthat encourages the formation of molecular complexes. In the first step,aniline monomers are absorbed onto a polyanion chain dissolved insolution. The resulting adduct, polyanion:(aniline)_(x) has signaturesthat can be monitored and verified. In the second step, the attachedaniline monomers are oxidatively polymerized to form the polymericcomplex.

[0020] The adduct of polyanion:(aniline)_(x) may take the shape of atight coil or extended chains. The shape of the adduct controls themorphology of the polymerized product. A tight-coiled adduct results inglobular polyaniline complex, while an extended chain adduct results inthin fibers of the double-strand complex aggregates (100 nm diameter×5micron length). Thus the polyanion functions as a template during thechemical synthesis, and the template becomes the second strand of the“double-strand” polyaniline after polymerization.

[0021] That is the template guided synthesis allows for controlling themorphology (e.g., fibrous or globular) of the complex as well as theconformation (e.g., coiled or extended chain, helical or sheetconformation) of the polymer. Because the polymer has delocalizedelectrons on the polymer backbone, the van der Waals and electrostaticinteraction of the polymer with the mobile phase can be quite differentfrom the conventional materials for stationary phase. This feature isadvantageous for separation of proteins, DNA, and drugs because thedelocalized binding between the polymer and the analytes can be designedto be specific enough to be selective among the molecules that areotherwise difficult to be separated.

BRIEF DESCRIPTION OF THE DRAWING(S)

[0022]FIG. 1A depicts electropherograms for iodide anions on an uncoatedcapillary.

[0023]FIG. 1B depicts electropherograms for iodide anions on a coatedcapillary.

[0024]FIG. 2A depicts electropherograms for bromide anions on anuncoated capillary.

[0025]FIG. 2B depicts electropherograms for bromide anions on a coatedcapillary.

[0026]FIG. 3A depicts an electropherogram for nucleotides at a pH of 6on a coated capillary.

[0027]FIG. 3B depicts an electropherogram for nucleotides at a pH of 6on an uncoated capillary.

[0028]FIG. 4A depicts an electropherogram for nucleotides at a pH of 7on a coated capillary.

[0029]FIG. 4B depicts an electropherogram for nucleotides at a pH of 7on an uncoated capillary.

[0030]FIG. 5A depicts an electropherogram of nucleotides (50 Mm acetatebuffer, at a pH of 4).

[0031]FIG. 5B depicts an electropherogram of nucleotides (20 Mm acetatebuffer, at a pH of 4).

[0032]FIG. 6A depicts an electropherogram of albumin and glyceraldehydeson a coated capillary.

[0033]FIG. 6B depicts an electropherogram of albumin and glyceraldehydesOn an uncoated capillary.

DESCRIPTION OF THE PREFERRED EMBODIMENTS(S)

[0034] In the preferred embodiment of the invention, the double strandedconductive polymer system, which is applied to or, alternatively,comprises the stationary phase of the chemical separation system, ispolyaniline: polymethylmethacrylate. The synthesis of polyaniline:polymethylmethacrylate (PANI: PMMA) disclosed herein is disclosed inInternational Application No. PCT/US96/11646, entitled “ElectroactivePolymer Coatings for Corrosion Control” and the same is incorporated byreference in its entirety into this disclosure.

[0035] Tempate-Guided Synthesis of Double-Strandpolyaniline:poly(acrylic acid-co-methylacrylate

Step I

[0036] The mole ratio between the carboxylic acid groups and the anilinemonomer units ranged from 2:1 to 1:1. The resulting polymeric complexwas soluble or dispersable in water, methanol and ethanol. The procedurefor synthesis of polyaniline:poly(acrylic acid) complex has beenreported supra and the reported procedures were followed.

Step II

[0037] The polyaniline:poly(acrylic acid) complex prepared in step 1 wasdissolved in methanol. To this solution is added catalytic amount ofbenzene suflonic acid or toluene sulfonic acid to serve as a catalystfor esterification reaction. The solution was refluxed for 3 days. Theesterification reaction converted some of the carboxylic acid group intothe methyl acetate group. This lowered the solubility of the complex inmethanol and the polymeric complex precipitated out of the solution. Theprecipitate was filtered out and dissolved in ethyl acetate. If a higherdegree of esterification was desired, the precipitate could beredissolved in 1:1 mixture of ethylacetate and methanol, and thesolution further refluxed until precipitate was again formed. Thisprecipitate was soluble in pure ethyl acetate but is not soluble 1:1mixture of ethyl acetate and methanol.

Coating Procedures

[0038] Two coating procedures where employed using the PANI: PMMA doublestranded conductive polymer.

Procedure 1

[0039] A dilute solution of the polymer in ethyl acetate was pushedthrough a fused silica capillary. (The capillary(s) used were-polyimidecoated fused silica 100 to 300 mm inner diameter). A small amount of thepolymer was sealed in a vial and the end of the capillary pushed throughthe seal. An air filled syringe was also pushed through the seal. Whenthe syringe was depressed the air displaced the polymer solution and thesolution flowed through the capillary. The polymer could be seen exitingthe other end of the capillary and so it was known that the solution wasbeing pushed through the capillary. After a few minutes, air was pushedthrough the capillary in the same manner to remove the excess polymerfrom the capillary.

Procedure 2

[0040] The second procedure was more exact. The concentration of thepolymeric solution was determined to be approximately 2.1 g/L. Thesolution was diluted in ethyl acetate to produce a solution that was0.1% in ethyl acetate. The capillary was pretreated (to clean thesurface) by washing it with 0.1M sodium hydroxide for 30 minutesfollowed by 0.1M hydrochloric acid for another 30 minutes. Finally, itwas rinsed with distilled water for 30 minutes. The capillary was thenheated in a nitrogen atmosphere at 100° C. for 2 hours to dry thecoating. The capillary was cooled before use. A comparison was made ofthe readings on the UV spectrometer (at 214 nm) after each stage of thecoating procedure and it was noted that the presence of the coatingcould be detected by-the spectrometer.

[0041] The invention will further be described with reference to thefollowing non-limiting examples.

EXAMPLE I Iodide and Bromide Anions

[0042] Initial tests of the coating were performed using iodide andbromide anions. These were chosen as the model compounds because theyare simple, charged compounds, which absorb in the UV region.

Separation Conditions Results

[0043] TABLE 1 Results for iodide and bromide anions IODIDE IODIDEBROMIDE BROMIDE pH 5.25 pH 6.75 pH 5.25 pH 6.75 UNCOATED 3.80 4.92 4.195.09 CAPILLARY (MIGRATION TIME minutes) COATED 10.83 7.71 9.16 8.01CAPILLARY (MIGRATION TIME minutes)

[0044] Referring to FIGS. 1A, 1B and Table 1, the coating was present onthe capillary surface as illustrated by the differences obtained on theuncoated and coated capillaries. An increase in pH was accompanied by anincrease in migration time for the bare capillary and a decrease inmigration for the coated capillary. The coating affects the elution ofanions in the capillary and the coating was changing with the pH of thesolution.

EXAMPLE II Monophosphate Nucleotides

[0045] The use of PANI: PMMA as a coating for a capillary in capillaryelectrophoresis is present and effective for the analysis of small,biological molecules. The following results reveal the effect of pH andbuffer concentration on the coated capillary and the effectiveness ofpre-treating the fused silica capillary as part of the coatingprocedure.

Separation Conditions

[0046] The capillaries were coated using procedure 2. The separationconditions were +20 kV, a 20 Mm acetate buffer, an injection time of 10s and the separation was performed on a Waters Quanta capillaryelectrophoreses system.

Results

[0047] TABLE 2 Electroosmotic flow comparison for coated and uncoatedcapillaries at a pH of 4 ELECTROOSMOTIC FLOW UNCOATED CAPILLARY 2.89 ×10⁻⁴ COATED CAPILLARY 6.31 × 10⁻⁴

[0048] TABLE 3 Migration time comparison for coated and uncoatedcapillaries at a pH of 4 MIGRATION TIME ON MIGRATION TIME ON COATEDCAPILLARY UNCOATED CAPILLARY CMP 11.45 minutes 5.30 minutes AMP 14.35minutes 5.45 minutes GMP 17.66 minutes 5.60 minutes UMP 19.89 minutes5.80 minutes

[0049] Referring to FIGS. 2A, 2B, Table 2 and Table 3, when the samenucleotide samples were run on both a PANI: PMMA coated capillary and anuncoated fused silica capillary under the same conditions, significantchanges in the separation were attained signaling the presence of thePANI: PMMA coating. The EOF values for the capillaries reveal that thecoated capillary is significantly faster than the uncoated capillary.Using a bare capillary it was not possible even under optical conditionsof high pH to separate monophosphate nucleotides in such a short amountof time. This constitutes a rapid analysis technique. The migrationwindow was also observed to be much shorter than that of the uncoatedcapillary.

[0050] Additional tests were performed to observe the influence of pH onthe coated capillaries. In both cases the separation attained on thecoated capillary is accompanied by the separation attained on theuncoated capillary.

Separation Conditions

[0051] The capillaries were coating using procedure 1. The separationconditions were +20 kV, a 50 Mm phosphate buffer, an injection time of10 s and the separation was performed on a Waters Quanta capillaryelectrophoresis system.

Results

[0052] TABLE 4 Migration time comparison for coated capillaries at a pHof 6 and a pH of 7 MIGRATION TIME ON MIGRATION TIME ON COATED CAPILLARYCOATED CAPILLARY at a pH of 6 at a pH of 7 CMP 18.20 minutes 18.00minutes AMP 19.51 minutes 16.31 minutes GMP 20.34 minutes 15.00 minutesUMP 21.00 minutes 19.12 minutes

[0053] TABLE 5 Migration time comparison for uncoated capillaries at apH of 6 and a pH of 7 MIGRATION TIME ON MIGRATION TIME ON UNCOATEDCAPILLARY UNCOATED CAPILLARY CMP 17.50 minutes 41.00 minutes AMP 16.00minutes 30.25 minutes GMP 18.22 minutes  5.60 minutes UMP 19.21 minutes38.27 minutes

[0054] Referring to FIGS. 3A, 3B, 4A, 4B, Table 4 and Table 5, themigration order differences of the nucleotides in the coated anduncoated capillaries revealed the presence of the coating and thedifference in behavior between the coating and fused silica. It can beseen that there is a large difference in migration times for thecapillaries at a pH of 7 and that the coated capillary presents a largeadvantage at this pH. The two capillaries were the same size and thedifference cannot be attributed to any feature other than the presenceof the coating.

[0055] Additional experiments were performed to reveal the what effectthe coated capillaries had at different concentrations of the samebuffer differences in the migration times of the monophosphatenucleotides are noted. The initial analysis was all performed with ahigh concentration buffer (50 mM) but later analysis was also performedat 20 Mm.

Separation Conditoins

[0056] The samples were run on the Water's Quanta capillaryelectrophoresis instrument. An injection time of 10 seconds and arunning voltage of 20 kV were used. Acetate buffer at the two differentconcentrations was prepared by the same method. The capillaryequilibrated at each concentration for about 40 minutes before thesamples were injected. TABLE 6 Migration time comparison for coatedcapillaries at a pH of 4 and different concentrations MIGRATION TIME ONMIGRATION TIME ON COATED CAPILLARY COATED CAPILLARY with 50 Mm bufferwith 20 Mm buffer CMP  9.25 minutes 5.30 minutes AMP 11.13 minutes 5.45minutes GMP 14.35 minutes 5.60 minutes UMP 15.12 minutes 5.80 minutes

Results

[0057] The coating can be seen to respond to the difference in bufferconcentration. The response was highly reproducible. It can be observedthat the lower concentration of buffer gives the faster analysis. It canbe concluded that at low pH the coating appears to produce excellentseparations of nucleotides. It enhances the separations attained by theuncoated capillaries considerably.

EXAMPLE III Proteins

[0058] Example II illustrate the ability of the coating to separate,small, biological molecules in a fast, efficient and reproduciblemanner. The following example illustrates the ability of the coating toseparate, large, biological molecules, such as proteins, in a fast,efficient and reproducible manner. A mixture of albumin andglyceraldehydes was prepared and analyzed.

Separation Conditions

[0059] The analysis was performed with a 20 Mm borate buffer at a pH of8, injection time of 20 seconds and an applied voltage of +15 kV.

Results

[0060] TABLE 7 Efficiency values for albuim on the coated and uncoatedcapillary EFFICIENCY ON EFFICIENCY ON COATED CAPILLARY UNCOATEDCAPILLARY ALBUMIN 179496 14652 THEORETICAL THEORETICAL PLATE NUMBERSPLATE NUMBERS

[0061] Coating capillary increased efficiency by one order of magnitude.

[0062] Referring to FIGS. 6A, 6B and Table 7 and in regard to theprotein analysis on the uncoated capillary, the protein appears to havenot eluted and is most likely adhered to the wall of the capillary.Using the coated capillary the protein is seen to elute with good peakshape and in a relatively short amount of time.

[0063] The foregoing description has been limited to a specificembodiment of the invention. It will be apparent, however, thatvariations and modifications can be made to the invention, with theattainment of some or all of the advantages of the invention. Therefore,it is the object of the appended claims to cover all such variations andmodifications as come within the true spirit and scope of the invention.

Having described our invention, what we now claim is:
 1. (Cancelled)
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 3. (Cancelled)
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 5. (Cancelled)
 6. (Cancelled)
 7. (Cancelled)
 8. (Cancelled)
 9. (Original) A method for separating components in a medium comprising: contacting a double stranded conductive polymer system to a stationary phase; and flowing the medium into a contacting relationship with the stationary phase to capture and separate the components.
 10. The method according to claim 9 wherein the double stranded conductive polymer system is comprised of a π-conjugated polymer and a polyelectrolyte.
 11. The method according to claim 10 wherein the π-conjugated polymer is selected from the group consisting of polyaniline, plypyrrole, polythiophene, and poly(phenylene vinylene).
 12. The method according to claim 10 wherein the polyelectrolyte is selected from the group consisting of poly(acrylic acid), poly(methylvinylether-co-maleic acid), poly(butadiene-co-maleic acid), poly(vinylsulfonic acid), poly(styrenesulfonic acid), poly(methacrylic acid), poly(L-glutamic acid), poly(L-Asparic acid), and poly (methylacrylate-co-acrylic acid).
 13. The method according to claim 9 wherein the stationary phase is selected from the group consisting of particulate materials, porous materials, semi-solid materials and solid materials.
 14. The method according to claim 9 wherein the stationary phase is a fused silica capillary.
 15. The method according to claim 9 wherein the medium is a liquid or gas.
 16. The method according to claim 9 wherein at least one of the components is a macromolecule. 